Plant growth strongly relies on local activity of the signalling molecule auxin, which is a small organic molecule regulating cell division, cell elongation and cell differentiation in plants. This molecule has great impact on the final shape and function of cells and tissues in all higher plants. Specificity in auxin responses is generated through the DNA-binding AUXIN RESPONSE FACTORs (ARFs) family. Until now, the exact mechanism of ARF activation at molecular level is still unknown. FRET-FLIM analysis of different ARF interaction combinations suggests the formation of homo- and hetero-oligomers. This is illustrated in Figure 1 where an overview of several full-length ARF interactions is shown. Currently, ARF homo- and heterodimerization is hypothesized to take place through domain (III/IV) located at the C-terminus.
However, recently, our department has developed a novel structure-based model for ARF protein action and DNA-binding specificity. Surprisingly, these in vitro assays showed that the DNA-binding domains (DBD’s) of ARF proteins require both specific protein-DNA interactions and protein dimerization to regulate their target genes. It has been shown that DBDs of ARF1 and ARF5 can form homodimers in absence and presence of DNA. However, it remains unclear about heterodimer formation of these DBDs and their biological role in live plants.
Several student projects are described that contribute to the overall aim of this research. These projects involve determination of binding constants of ARF-ARF and ARF – DNA interactions, quantification of ARF concentrations in Marchantia polymorpha (oldest land plant) or protein turnover rates. The outcome of this research will be the starting point for a chemical screen to identify compounds that alter ARF-ARF and ARF-DNA interactions and the development of a mathematical model that identify critical factors within the protein network that affect signal output.
A) The DNA binding domain of ARF5 is coupled to either CFP (donor) or YFP (acceptor). Large distance between CFP and YFP results in low FRET whereas high FRET signals are obtained if ARF domains are in close proximity and correct orientation.
FRET can be quantified by fluorescence lifetime analysis. A fluorescence intensity image (B) is showing the nuclear expression of the donor ARF5-sCFP3A in a plant protoplast. Per pixel the time resolved photon distribution is plotted (C) of which the fluorescence lifetime is calculated and shown as a false colour-coded fluorescence lifetime image (D). Interaction of a donor with an acceptor molecule results in a decrease of the fluorescence lifetime (C).
Student project no. 1; AtARF5
What is the binding constant of ARF5-DBD homodimers?
A construct encoding ARF5 DBD with an unnatural amino acid located at the specific position (RWP) or at the C-terminus has been prepared. This project involves the production of these constructs and label these unnatural amino acids with fluorescent dyes using click chemistry.
Techniques; biochemical assays, protein production and purification from E. coli, FRET analysis.
Student project no. 2; AtARF5
Determination of binding constant of ARF5-DBD using fluorescence spectroscopy
This project involves the quantification of the binding constants of ARF5-DBD using fluorescence correlation spectroscopy. C-terminal fluorescently tagged ARF5-DBD constructs are available but needs to be produced and purified
Techniques; biochemical assays, protein production and purification from E. coli, FCS measurements.
Student project no. 3; AtARF5
What is the biological function of ARF5 and ARF1 homo- and hetero-dimerization?
1. Construct preparation for generation of A. thaliana stable transgenic lines expressing:
c. pARF1::ARF1- YFP,
d. pARF5::ARF5-YFP and pARF5::ARF5-CFP,
e. pARF5::ARF5-YFP and pARF1::ARF1-CFP
2. Agrobacterium-mediated transformation of mp mutant and screening for transgenic lines
3. Phenotype analyses of pARF5::ARF5-YFP, pARF5::ARF5-CFP expressing plants to validate the biological activity of constructs
4. Chemical treatment of transgenic lines with compounds modulating ARF homo- hetero-dimerization and evaluating their effect on:
a. homo- hetero-dimerization efficiency
b. ARF localization
c. ARF expression level
d. overall plant growth
Student project no. 4; MpARF1,2,3
Quantification of Marchantia polymorpha ARF interactions
This project concerns the cloning and expression of MpARF, either full length or MpARF-DBD. Marchantia has only three ARF proteins to execute auxin mediated signalling. We aim to produce all three MpARFs as well as the receptor for auxin and repressor protein. In the end, a network of protein interactions will be established for the development of mathematical model describing auxin mediated signalling.
Techniques; molecular biology, biochemical assays, protein production and purification from E. coli.